Project Summary Iris muscle is a rare example of a neural tube derived muscle and surprisingly little is known how this remarkable break in germ layer rules is carried out. While we know PAX6 expression plays a role in iris muscle specification and that ACTA2 is present, how iris muscle cells are specified at the optic cup tip is not known. The long-term goal is to understand the signaling events that activate the muscle fate program from the developing optic cup. The sequence of fate decisions that enable this rare example of muscle differentiation from developing neural tube is the process this proposal intends to examine and the objective of this proposal. We predict many of the candidates necessary for smooth muscle cell differentiation will be involved, but there will be distinct processes necessary for iris muscle to develop that is unique, due to their specification from the neural tube instead of neural crest or mesoderm. The retina when stimulated with inflammatory cytokines differentiates towards muscle cell fate, albeit pathologically in diseases such as Proliferative Vitreoretinopathy (PVR), Proliferative Diabetic Retinopathy, Age-related Macular Degeneration Macular Pucker and more. Adult human RPE (ahRPE), retinal glia and retinal pericytes express contraction apparatus, leading to the development of myocontractile membranes, which upon contraction, cause retinal detachment and vision loss. The central hypothesis is the ability of the retina to transdifferentiate into a muscle phenotype originates from having a shared developmental origin with the iris muscle. The rational underlying this proposal is: iris muscle development is not well known and understanding iris muscle development may provide insight into how the retina develops into contractile membranes in eye diseases. The central hypothesis will be tested by pursuing these Specific Aims: 1) To test whether muscle associated genes OLFM2 and MYOCD identified in patient dissected contractile membranes are necessary for iris muscle differentiation in an eye organoid model. 2) To compare the adult human iris muscle gene signature with expression of human eye organoids by single cell RNA-seq. 3) To confirm role of OLFM2 and MYOCD by evaluating existing KO mice. The iris muscle differentiates at the tip of the optic cup, a continuous bilayer with RPE, developed from the neural tube. We will pursue these aims using an innovative combination of analytical and experimental techniques. These includes using our previously established protocol to isolated RNA from human adult cadaver tissue with sufficient survival and yield to enable single cell RNA-seq. Moreover, we have developed an eye organoid differentiation protocol that generates presumptive iris muscle cells, a first. The research proposal is significant, because the results will begin to describe factors participating iris muscle development and we will gain insight into contractile membrane formation in eye disease. The expected outcome of this work is a more complete understanding of neuroectoderm derived muscle specification and contractile membrane formation. The results will have a positive impact as the new insight gained will point to new targets for the prevention of vision loss.